A novel "pearl box" cataract associated with a mutation in the connexin 46 (GJA3) gene.

Purpose To undertake mutation screening in the connexin 46 (GJA3) gene in seven congenital cataract families of Indian origin. Methods Seven Indian families with congenital cataract were analyzed by detailed family history and clinical evaluation. Each family had two to five affected members. Mutation screening was carried out in the candidate gene, connexin 46 (GJA3), using bidirectional sequencing of amplified products. Segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP). Results Sequencing of the coding region of GJA3 showed the presence of a novel, heterozygous C260T change in one family (CC-472) who had two affected members. The cataract phenotype gave the appearance like a "pearl box" in these two affected individuals of this family. The observed C260T substitution created a novel restriction enzyme site for NlaIII and resulted in substitution of highly conserved threonine at position 87 by methionine (T87M). NlaIII restriction digestion analysis revealed this nucleotide change was not in unaffected members of this family or in 100 unrelated control subjects (200 chromosomes) with the same ethnic background. Conclusions This is a novel mutation identified in the second transmembrane domain of the connexin 46. These findings thus expand the mutation spectrum of the GJA3 in association with congenital cataract.

viously published primer sequences [11]. Genomic DNA from two affected and one unaffected individual from each family were amplified. PCR and sequencing reactions were performed following conditions detailed elsewhere [12,13]. Electrophoresis of purified sequencing reaction products was performed on 5% urea-polyacrylamide gel on ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA), and data was analyzed using sequence analysis software version 3.4.1 (Applied Biosystems).
Restriction endonuclease analysis: The DNA fragment harboring the mutation was amplified for both affected and unaffected family members, and PCR products were digested with NlaIII restriction enzyme following directions given by the manufacturer (New England Biolabs, Beverly, MA). Re- striction digestion products were analyzed on a 2.5% agarose gel. In addition, 100 unrelated controls from similar ethnic background were also similarly tested.

RESULTS
In the present study, four families with autosomal-dominant congenital cataract and three families with autosomal-recessive congenital cataract were investigated. Each family had 2-5 affected members with different clinical morphologies (one family each with pearl box, posterior cortical, granular, absorbed membranous, and round ball cataract and two autosomal-recessive families with posterior subcapsular type cataract). Mutation analyses of the connexin 46 (GJA3) gene in these seven families revealed a novel heterozygous mutation in only one family (CC-472). In this family, a son and his mother were affected with bilateral congenital cataract (Figure 1A). The opacities involved both the embryonal and fetal nuclei separated by a clear space. Fine white spots made up the opacity of the fetal nucleus ( Figure 1B). The embryonal nucleus was composed of coarse white spots of various sizes that were round when located toward the center and linearshaped in the periphery. There was an optically empty space between the embryonal nuclear and fetal nuclear opacities. The peripheral zone outside the fetal nucleus was clear. The cataract phenotype gave the appearance of a box produced by the fetal nuclear opacities and pearls by the embryonal nuclear opacities ( Figure 1C). Hence, this phenotype was termed "pearl box" cataract.
Direct sequencing of GJA3 revelaed a novel heterozygous C>T transition ( with methionine at codon 87 (Thr87Met). This substitution created a novel NlaIII restriction site that segregated completely with the disease phenotype in this family ( Figure 2C). This nucleotide change was not observed in the 100 control subjects from similar ethnic background tested by restriction digestion analysis (data not shown). Testing of six additional families revealed no disease-linked change or any polymorphic change in GJA3.

DISCUSSION
We report a novel change, T87M in the connexin 46 gene, in one family (CC-472) who has what we term "pearl box" cataract. The T87M substitution is likely to be disease causative as it segregated among the affected members only and was not detected either in unaffected family members or in 100 unrelated controls. The 260C>T substitution observed in the present study resulted in the replacement of polar amino acid threonine (T) with nonpolar amino acid methionine (M) at codon 87 (T87M) localized in the second transmembrane domain (M2) of the connexin 46 polypeptide (Figure 3). This is the first identified mutation that lies within in the second transmembrane domain (M2) of the connexin 46 polypeptide associated with novel "pearl box" cataract.
A multiple sequence alignment of the amino acid sequences of connexin 46 showed that threonine at position 87 is phylogenetically conserved in the second transmembrane domain of connexin 46 in different species ( Figure 4A) and also in different human α-connexins ( Figure 4B). This indicates that the threonine at position 87 in connexin 46 is likely to be functionally important and the T87M mutation may therefore has a detrimental physiological effect. In the connexin 50 (GJA8) gene localized at 1q21, corresponding mutations (P88S and P88Q) in the second transmembrane domain (M2) resulting in zonular pulverulent [14] and lamellar pulverulent cataract [15], respectively, have been reported. Pal et al. [16] further reported that P88S mutant connexin 50 abolishes the func- tion of the gap junction channel and acts in a dominant negative manner. In humans, P87S and P87A mutations located within M2 of the connexin 32 (GJB1) gene have been reported in association with Schwann cell dysfunction and peripheral nerve degeneration in X-linked Charcot-Marie-Tooth disease [17][18][19]. The authors hypothesized that these mutations (P87S and P87A) in M2 of the connexin 32 polypeptide may impair voltage-dependent opening and closing of gap junctions in electrically excitable tissues. Suchyna et al. [20] have also demonstrated that in the connexin 26 polypeptide proline at codon 87 has a function in voltage-gating of gap junctions. Although, the effect of T87M mutation observed in the present study on the activity of the connexin 46 has not been directly tested, we speculate that, like P88S in the GJA8 and other dominantly inherited mutations reported in different connexins, this mutation also results in inappropriate association of connexins and alters the function of endogenous wild-type connexins in the affected individuals in a dominant negative way.
Defects in the connexin 46 and connexin 50 genes have been reported to cause cataract in mice. Point mutations A47C and V64A in the connexin 50 gene have been reported to result, respectively, in nuclear opacities (No2) and nuclear with posterior suture opacities (Aey5) in mice [21,22]. Gong et al. [23] reported that mice homozygous for disrupted connexin 46 developed nuclear cataracts due to failure in maintenance of differentiation and of crystallins solubility, while connexin 50 knockout mice had reduced ocular growth along with nuclear cataract [24,25].Targeted replacement of connexin 50 with connexin 46 in mice has revealed the role of connexin 50 in lens and eye growth and that of connexin 46 in maintaining differentiation by nonspecific restoration of intercellular communication [26].
The cataract phenotypes that are reported to be linked with the GJA3 mutations share genotype-phenotype similarities to some extent, but they also exhibit some differences with respect to the appearance and location of opacities within the lens. At this point, 12 mutations in GJA3 have been reported to be associated with autosomal-dominant congenital cataract in humans involving different domains of connexin 46 polypeptide ( Table 1)  Alignment data indicate that threonine is highly conserved in different species (A) and in different human alpha-connexins (B). Arrow points to the conserved threonine at position 87, which is marked in red.

TABLE 1. REPORTED MUTATIONS IN GJA3 ASSOCIATED WITH DIFFERENT CONGENITAL CATARACT PHENOTYPES IN DIFFERENT FAMILIES
Mutation spectrum of the connexin 46 (GJA3) gene and cataract phenotypes in different congenital cataract families. 801 family) is different in its appearance from the earlier reported types (Table 1) as it appears like pearls in a box ( Figure 1B,C). The differences in the morphologies of cataract phenotypes associated with mutations in the GJA3 in different families may be attributed to the action of modifier genes or environmental factors that could affect the expression of the connexin 46 gene and hence resulting cataract types.
In summary, we describe a novel heterozygous T87M mutation in the connexin 46 polypeptide associated with "pearl box" cataract. On the basis of observed phenotypic as well as genotypic variability as compared to previously published reports, the present study further expands the genetic and phenotypic heterogeneity of congenital cataract.